Compositions for human and animal consumption containing reduced folates and methods for making and using same

ABSTRACT

A composition for human or animal consumption for supplying folate which includes a natural isomer of reduced folate, such as (6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolic acid, and their polyglutamyl derivatives is disclosed. Such compositions include multivitamin preparations (with or without minerals and other nutrients); breakfast foods such as prepared cereals, toaster pastries and breakfast bars; infant formulas; dietary supplements and complete diet and weight-loss formulas and bars; animal feed (for example pet foods) and animal feed supplements (such as for poultry feed). The amount of the natural isomer of a reduced folate in a composition for human consumption can range between about 5% and about 200% of the daily requirement for folic acid per serving or dose.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/010,898, filed Jan. 31, 1996.

FIELD OF THE INVENTION

The present invention relates generally to the field of nutrition, andmore specifically to food and vitamin preparations containing thenatural isomer of reduced folates.

BACKGROUND OF THE INVENTION

The folates are ubiquitous to nearly all forms of life. Humans and manyother animals lack the capacity to make their own folate which thus isan essential vitamin, one type of essential nurient. Anemia especiallyduring pregnancy and in the geriatric population was an early indicationof a dietary requirement for folate. A major function of folate is toremove one-carbon units from molecules being metabolized and thendeliver them to molecules being synthesized. As an example. folateparticipates in the formation of the nucleic acids. Further, theactivity of DNA is controlled, in part, by methylation, and the primarymethylating agent of the body (S-adenosylmethionine) is made in ametabolic cycle involving a folate. Many studies have, therefore,focused on the relationship of folate status to cancer susceptibility,especially colorectal adenoma.

The importance of folate to proper growth is clearly evident in theoccurrence of neural tube defects in newborn infants. Reports fromseveral countries have shown that a majority of such cases areassociated with low folate levels in the mother. The incidence of thesedefects as well as of cleft lip/palate is considerably reduced whenwomen are given folic acid (I) starting early in pregnancy. Recently, asignificant correlation has been discovered between vitamin deficiency,especially of folate, and peripheral vascular disease, a major cause ofdeath. A high percentage of individuals with this affliction haveabnormal blood levels of homocysteine, a precursor to methionine in thefolate dependent step of the S-adenosylmethonine cycle. Folatedeficiency has also been linked to defective maturation of a number ofdifferent cell types, to nervous system disorders, and to decreasedimmune response.

The clear relation of folate intake to health has caused manygovernmental agencies around the world (such as the U.S. NationalReasearch Council) to specify a recommended dietary allowance ("RDA")for folate. In the U.S. these values are used by the Food and DrugAdministration to establish the Reference Daily Intake ("RDI") that islisted on food labels, currently 0.4 mg for adults. The highest dailyamount of folic acid recommended by a country is 2.0 mg for healthyadults. Many products are available that contain RDI or near RDI levelsof folic acid (I) including most daily multiple vitamins. These can bepurchased in solid (eg. tablet, capsule, or powder) or in liquidformulations, both over-the-counter and by prescription. In the U.S.folic acid (I) is also available by itself typically at a dosage of 0.4mg, but also up to 0.8 mg in health food stores. Many complete diets,infant diets, dietary supplements and weight loss products also containfolic acid (I). In some countries folic acid is added to specific foodtypes as determined by health officials to provide adequate folate tothe general population without risking excess consumption. Manybreakfast foods, such as cereals, cereal bars, breakfast drink mixes,breakfast bars and toaster pastries have folic acid (I) added at amodest fraction of the RDI, typically 10-50% of the adult value perserving. In many of these uses the folic acid (I) is accompanied byother vitamins, sometimes at RDI dosages, but also at lower or muchhigher levels. Frequently, though not always, essential mineralnutrients are also present. Further, many products also includecompounds hypothesized to have health related value, but which eitherhave not been officially recognized as effective, or for which optimalamounts have not been set. Products such as those described above aremeant to fill an important and wide spread need for folate, especiallyamong those whose dietary habits would otherwise preclude intake of asufficient amount of this vitamin.

Folic acid (I) is a component of many animal and pet foods. It is alsoincluded in powders or liquids used as animal feed supplements, often incombination with other nutrients. For example, the National ResearchCouncil (NRC) recommends diets containing 0.2 mg and 1.0 mg of folicacid (I) per kg of dry diet (assuming 5 kcal metabolizable energy pergram) for dogs and cats, respectively. For chicks the NRC hasrecommended 0.55 mg folic acid per kg of diet, although recentliterature suggests that the optimal value is about three times higherthan this.

The form of folate currently added to all commercial vitaminpreparations or which is added to foods, folic acid (I) (also known aspteroyl-L-glutamic acid), is not one of the major forms found in naturalfresh foods. The structure of folic acid (I) differs from the mostabundant natural folate in several aspects. First, the side-chain ofnatural folates in almost all fresh foods contains more than oneL-glutamic acid moiety. Frequently, five to seven (but covering aconsiderable span of more or fewer) of this amino acid are linkedtogether into a polyglutamate chain. It is well known, however, that theprimary form by which folates are absorbed has only a single glutamateresidue. Cleavage of the extra glutamates of dietary folates is usuallyaccomplished by an enzyme in the digestive tract. In this aspect folicacid (I) is not at a disadvantage in comparison to naturally occurringfolates.

The second difference between folic acid (I) and natural folates is thatwhereas the pteridine ring of the former (I) is fully oxidized, naturalfolates in fresh uncooked foods are mostly present as the tetrahydroforms. Almost all of the known physio-logical functions of folate areperformed by tetrahydrofolic acid, (6S)-FH₄ (II), or by a one carbonderivative of it illustrated as follows: 5-methyl-(6S)-FH₄ (III),5-formyl-(6S)-FH₄ (IV), 10-formyl-(6R)-FH₄ (V), 5,10-methylene-(6R)-FH₄(VI), 5,10-methenyl-(6R)-FH₄ (VII), and 5-formimino-(6S)-FH₄ (VIII). Thestructural formula for each of these compounds is provided below.##STR1## There is no known direct cofactor function for folic acid (I)itself in humans. Some (6S)-tetrahydrofolic acid polyglutamate is foundin plants or animals, but the majority of folate is polyglutamate formsof either 5-methyl-, 5-formyl-(6S)-tetrahydrofolic acid, and in somecases 10-formyl-tetrahydrofolic acid. Presumably, most of the folic acid(I) found in biological food sources results from oxidation, especiallyon storage. When folic acid (I) is absorbed by the digestive tract it iseventually reduced to active (6S)-tetrahydrofolic acid (II) by theenzyme dihydrofolate reductase.

The oral bioavailability of folic acid (I) has been shown to be widelyvariable. The literature contains reports of individuals having poorintestinal uptake of folic acid (I) who respond normally tointramuscular injection of folic acid (I), or had normal serum folatestatus prior to any folic acid challenge. Several small scaleinvestigations in which the values have been averaged have concludedthat the oral uptake of several of the reduced folates is similar tofolic acid (I). However, there is reason to believe that a segment ofthe population possesses adequate oral response to reduced folates, butnot to oral folic acid (I).

5-Formyl-tetrahydrofolic acid (also known as leucovorin or folinic acid)has long been used in therapeutic doses for several diseases. Examplesinclude rescue from the toxicity of methotrexate chemotherapy, and thesynergistic combination with fluorouracil for treatment of variouscancers. It is also given to treat acute anemia not due to B₁₂deficiency. 5-Methyl-tetrahydrofolic acid in high doses (for example, 50mg/day) has been patented for treatment of depression (and otherneurological disorders) (EP382019 and EP388827 to Le Grazie 1990, andEP482493 to Le Greca 1992).

That reduced folates have been overlooked as an improved source forproviding the RDA level is in part due to the stereochemistry of thesecompounds. In addition to the single chiral center of the L-glutamatechain in folic acid (I), the tetrahydrofolates contain a secondstereochemical center at carbon-6. Chemical reduction of folic acid (I)produces a nearly racemic mixture of the two isomers at this position.This is in contrast to the reduced folates found in nature which allconsist of a single diastereoisomer, all having the same L-configurationat carbon-6. (Compounds II--VIII are shown as the natural isomer). Formany years only the racemic 6(R,S) mixture of 5-formyl-tetrahydrofolicacid (leucovorin) has been used for therapy of diseases. Recently,however, concern over the possible effects of the unnatural isomercomponent has resulted in the commercial introduction of the purenatural isomer for these high dose disease treatments by Lederle,although at very high cost. Most therapeutic regimes utilizingleucovorin last a few weeks or perhaps months. The effect of a long termexposure to the unnatural isomer of reduced folates is unknown. Forexample, although little 5-formyl-(6R)-tetrahydrofolic acid is absorbed,there is considerable uptake of the unnatural isomer of5-methyl-tetrahydrofolic acid by the intestinal tract and other cells ofthe body which with continuous intake may lead to adverse consequences.

Until recently, processes for making the natural isomer of reducedfolates have been limited in scale, or costly, or both. These includechromatographic separation, enzymatic reduction, and fractionalcrystallization. The use of reduced folates as a daily source of vitaminrequires a method that is applicable to large scale production of thenatural isomer having high purity at a cost that will not place a burdenon the average consumer.

SUMMARY OF THE INVENTION

The present invention relates to a composition which includes one ormore natural isomers of reduced folate and a nutritional substance. Theone or more natural isomers of reduced folate is selected from the groupconsisting of (6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolicacid, 5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolicacid, 5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof. The nutritional substance is a food preparation, an essentialnutrient preparation, or a combination thereof. When the nutritionalsubstance is a food preparation, the food preparation includes two ormore food components. Each gram of the food preparation has a naturalmolar amount, N, of the one or more natural isomers of reduced folate, Nbeing greater than or equal to zero, and each gram of the compositionhas a total molar amount, T, of the one or more natural isomers ofreduced folate greater than N. When the nutritional substance is anessential nutrient preparation, the essential nutrient preparationincludes a vitamin other than ascorbic acid.

The present invention also relates to method for increasing the folatecontent of a nutritional substance. The method includes providing anutritional substance selected from the group consisting of a foodpreparation, an essential nutrient preparation, and combinationsthereof. The method further includes incorporating into the nutritionalsubstance a molar amount of one or more natural isomers of reducedfolate selected from the group consisting of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof. When the nutritional substance is a food preparation, the foodpreparation comprises two or more food components. When the nutritionalsubstance is an essential nutrient preparation, the essential nutrientpreparation comprises a vitamin other than ascorbic acid.

A significant number of people are folate deficient; especiallyvulnerable are those whose life style does not include sufficient freshfood sources of folates. An object of this invention is nutritionalcompositions in which the natural isomer of tetrahydrofolic acid, or aderivative thereof, is substituted for the usual folic acid (I) for thesatisfaction or partial satisfaction of the dietary requirement for thisvitamin. While some may not be greatly affected by the inclusion ofreduced folates in multivitamin preparations and breakfast foods, stilla substantial number of people, and thus the average health of thepopulation, will be improved by addressing the needs of those for whomfolic acid (I) bioavailability is poor. Consumer confidence with regardto consumption of a food or other nutritional product will be increasedwith the knowledge that the folate content is chemically identical tothe most abundant natural forms of this vitamin, except for theadvantageous absence of multiple glutamate residues. A further advantageis that health agencies will be aided in recommending optimal levelswhen a more uniformly absorbed form of folate is widely used.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition which includes one ormore natural isomers of reduced folate and a nutritional substance.Natural isomers of reduced folate suitable for use in the presentinvention include, for example, (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid, and5-formimino-(6S)-tetrahydrofolic acid. Other natural isomers of reducedfolate suitable for use in the present invention include thepolyglutamyl, such as the diglutamyl, triglutamyl, tetraglutamyl,pentaglutamyl, and hexaglutamyl, derivatives of (6S)-tetrahydrofolicacid, 5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolicacid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid, and5-formimino-(6S)-tetrahydrofolic acid. Any or all of the natural isomersof reduced folate can be present in its chirally pure form, or,alternatively, the composition can optionally contain a molar amount ofone or more unnatural isomers of reduced folate, such as(6R)-tetrahydrofolic acid, 5-methyl-(6R)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6S)-tetrahydrofolic acid,5,10-methenyl-(6S)-tetrahydrofolic acid,5-formimino-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof. The molar amount of the natural isomer of reduced folate can beequal to the molar amount of its corresponding unnatural isomer (aswhere the unnatural and natural isomer are present as a racemicmixture), or, preferably, the natural isomer of reduced folate can bepresent in a molar amount greater than the molar amount of thecorresponding unnatural isomer. The total molar amount of the one ormore natural isomers of reduced folate present in the composition can bebetween 5% and 200% of a human daily requirement for folate per acustomarily consumed quantity of the composition. As used herein, thetotal molar amount of the one or more natural isomers of reduced folateincludes natural isomers of reduced folates which are naturally presentin the nutritional substance as well as natural isomers of reducedfolates which might have been added to the nutritional substance. Thecustomarily consumed quantity of various compositions depends, ofcourse, on the nature of the composition. Where the composition includesa food preparation, the customarily consumed quantity is the amount ofthe food preparation customarily consumed per eating occasion, forexample, as set forth by the U.S. Food and Drug Administration for thepurpose of establishing realistic and consistent serving sizes for usein food labeling. Examples of customarily consumed quantities forvarious food groups can be found in 21 C.F.R. § 101.12, which is herebyincorporated by reference.

The human daily requirement for folate varies from person to person,depending on factors such as body weight, age, health, sex, and thelike. Suitable values for the human daily requirement for folate includeRDI and RDA values, promulgated, respectively, by the FDA and theNational Research Council ("NRC"). Presently, RDI values, expressend interms of micrograms of folic acid (nanomoles of folic acid), are 400 μg(907 nmoles) for adults and 800 μg (1814 nmoles) for pregnant women. Inview of this, the compositions of the present invention can have between45 and 1814 nnmoles or between 91 and 3625 nmoles of folate percustomarily consumed quantity of the nutritional substance. Current RDAvalues are published in National Research Council: Recommended DailyAllowances, 10th ed., Washington, D.C. (1989), which is herebyincorporated by reference. They are, again expressend in terms ofmicrograms of folic acid (nanomoles of folic acid), 25 μg (57 nmoles)for infants 0-6 months of age, 35 μg (79 nmoles) for infants 6 months toone year of age, 50 μg (113 nmoles) for infants 1-3 years of age, 100 μg(227 nmoles) for infants 7-10 years of age, 200 μg (454 nmoles) for maleadults, 180 μg (408 nmoles) for female adults other than pregnant adultfemales, and 400 μg (907 nmoles) for pregnant females. In view of this,typical compositions of the present invention can have between 2.8 and113 nmoles, between 4 and 159 nmoles, between 28 and 227 nmoles, between11 and 454 nmoles, between 22 and 907 nmoles, between 20 and 816 nmoles,or between 45 and 1812 nmoles of folate per customarily consumedquantity of the nutritional substance. Suitable values for the humandaily requirement for folates are also established by the World HealthOrganization as 7.03 nmoles/kg of body weight. For pregnant women thevalue calculated based upon body weight should be increased by about 454to about 680 nmoles.

The total molar amount of the one or more natural isomers of reducedfolate present in the composition can, alternatively, be between 5% and3000% of an animal daily requirement for folate per a customarilyconsumed quantity of the nutritional substance. The animal, whose dailyrequirement for folate is referred to above, can be, for example, a dog,a cat, a chicken, a cattle, a domestic animal, a goat, a horse, a mink,a fox, a sheep, or a swine. Suitable values for the an animal's dailyrequirement for folate are promulgated, for example, by the NRC inNutrient Requirements of Domestic Animals (Washington:National AcademyPress), particularly in those publications having the followingsubtitles: "Nutrient Requirements of Beef Cattle," Seventh RevisedEdition (1996, ISBN 0-309-05426-5); "Nutrient Requirements of Cats,"Revised Edition (1986, ISBN 0-309-03682-8); "Nutrient Requirements ofDairy Cattle," Sixth Revised Edition, Update (1989, ISBN 0-309-03826-X);"Nutrient Requirements of Dogs," Revised (1985, ISBN 0-309-03496-5);"Nutrient Requirements of Fish," (ISBN 0-309-04891-5); "NutrientRequirements of Goats: Angora, Dairy, and Meat Goats in Temperate andTropical Countries," (ISBN 0-309-03185-0); "Nutrient Requirements ofHorses," Fifth Revised Edition, (1989, ISBN 0-309-03989-4); "NutrientRequirements of Laboratory Animals," Fourth Revised Edition (1995, ISBN0-309-05126-6); "Nutrient Requirements of Mink and Foxes," SecondRevised Edition, (1982, ISBN 0-309-03325-X); "Nutrient Requirements ofPoultry," Ninth Revised Edition, (1994, ISBN 0-309-04892-3); "NutrientRequirements of Sheep," Sixth Revised Edition, (1985, ISBN0-309-03596-1); and "Nutrient Requirements of Swine," Ninth RevisedEdition, (1988, ISBN 0-309-03779-4) (collectively referred to as "NRCAnimal Nutrient Requirements"), which are hereby incorporated byreference.

As indicated above the nutritional substance can be a food preparationor an essential nutrient preparation. Essential nutrient preparationsare materials which contain one or more essential nutrients. Where onlyone essential nutrient is present in the essential nutrient preparation,that essential nutrient can be a vitamin other than ascorbic acid. Theessential nutrient preparation can, alternatively, include a vitaminother than ascorbic acid and, in addition, ascorbic acid. As usedherein, essential nutrients are those nutients which are required tosustain health but which cannot be effectively produced by one or moreanimals or by humans. Examples of essential nutrients are compiled in anumber of published sources, including Modern Nutrition in Health andDisease, 8th ed., Shils et al., eds., Philadelphia:Lea and Febiger(1994), which is hereby incorporated by reference. Essential nutientsare meant to include essential vitamins and provitamins thereof,essential fats, essential minerals, such as those minerals for whichdaily values have been recommended, and essential amino acids. Oneexample of an essential nutrient preparation is a formulation whichcontains a vitamin and a caloric content of less than 2.5 cal per drygram, preferably less than 2 cal per dry gram, most preferably less than1.8 cal per dry gram. Essential nutrient preparations also include thosematerials which contain at least one vitamin in an amount greater than15%, preferably greater than 20%, more preferably greater than 40% ofthe U.S. adult RDA for that essential nutrient per gram of essentialnutrient preparation. Still other suitable essential nutrientpreparations contain at least two vitamins, each in an amount greaterthan 10%, preferably greater than 15%, more preferably greater than 20%of the U.S. adult RDA for that essential nutrient per gram of essentialnutrient preparation. Suitable essential nutrient preparations arecommonly referred to as dietary supplements, vitamin supplements, andmineral supplements, multiple vitamin supplements, and the like, and aretypically available commercially in the form of pills, tablets,capsules, powders, syrups, and suspensions. Preferably, the essentialnutrient composition contains at least one essential nutrient in anamount greater than 25%, more preferably greater than 50%, and mostpreferably greater than or equal to 100% of the daily requirement forthat essential nutrient per customarily consumed quantity of theessential nutrient preparation.

As indicated above, the nutritional substance can also be a foodpreparation. Food preparations are materials which contain one or moreamino acid, carbohydrate, or fat, which are suitable for human or animalconsumption, and which are not essential nutrient preparations. It ispreferred that the food preparation be a two or more component foodpreparation. For example, a two or more component food preparation canbe a mixture of two or more one-component foods. One component foods arefoods which are derived substantially from a single natural source. Asmall percentage of the one-component food can be derived from a secondnatural source, but that percentage, by weight, is preferably less than5%, more preferably less than than 1%, more preferably less than 0.1%.One component foods include, for example, sugar, unsweetened juice,nectar, or puree from a single species of plant, such as unsweetenedapple juice (including a blend of different varieties of apple juice),grapefruit juice, orange juice, apple sauce, apricot nectar, tomatojuice, tomato sauce, tomato puree, and the like. Grain plants of asingle species and materials produced from grain plants of a singlespecies, such as corn syrup, rye flour, wheat flour, oat bran, and thelike are also considered to be one component foods. Alternatively, thetwo or more component food preparation can be a mixture of one or moreone component foods and one or more essential nutrients. Preferably, theamount of at least one of the one or more essential nutrients present inthe two component food is greater than the amount of the at least oneessential nutrient that is naturally present collectively in the one ormore one component foods. For example, where the essential nutrients arevitamin X and vitamin Y and where the one component food is orange juiceand where the orange juice naturally contains vitamin X and vitamin Y inamounts "Nx" and "Ny", respectively, it is preferred that thecomposition contain vitamin X and vitamin Y in amounts "Tx" and "Ty",respectively, so that Tx is greater than Nx, Ty is greater than Ny, orboth.

Food preparations particularly well suited to the practice of thepresent invention include breakfast foods, such as prepared cereals,toaster pastries, and breakfast drink mixes; infant formulas; dietarysupplements; complete diet formulas; and weight-loss preparations, suchas weight-loss drinks and weight-loss bars.

The food preparation can be one which naturally contains no naturalisomer of reduced folate. Alternatively, it can contain a natural molaramount of a natural isomer of reduced folate. For purposes of thisapplication, the molar amount of natural isomer of reduced folate (i.e.,collectively, the number of moles of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof) contained per gram of food is designated "N". For some foods,the molar amount of natural isomer of reduced folate naturally presentis known. For others, the molar amount of natural isomer of reducedfolate can be determined by a number of sensitive and specific methods,such as microbial growth dependence, folate binding protein basedassays, high-performance liquid chromatography ("HPLC") and gaschromatography ("GC"). Suitable methods are described, for example, inCossins, E. A., "Folates in Biological Materials," in Folates andPterins, Vol. 1, Blakley et al., eds., New York:John Wiley & Sons, pp.1-60 (1984), which is hereby incorporated by reference.

The molar amount of natural isomer of reduced folate present in thecomposition of the present invention is greater that the molar amount ofnatural isomer of reduced folate present in the food preparation. Forpurposes of this application, the molar amount of natural isomer ofreduced folate (i.e., collectively, the number of moles of(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof) contained per gram of composition is designated "T". Thus, Tnecessarily must be greater than N. Preferably, T/N is greater than105%; more preferably, it is greater that 110%; and, most preferably, itis greater than 120%.

As indicated above, the composition can, optionally, include one or moreunnatural isomers of reduced folate. When present in the composition,the one or more unnatural isomers of reduced folate is preferablypresent in a molar amount which is less than T minus N.

The present invention also relates to a method for increasing the folatecontent of a nutritional substance. The nutritional substance can be afood preparation, an essential nutrient preparation, or a combination ofa food preparation and an essential nutrient preparation. The folatecontent of the nutritional substance is increased by incorporating oneor more natural isomers of reduced folate into or with the nutritionalsubstance. This can be achieved by methods well known in the art of foodand essential nutrient preparation, such as by homegenizing, coating,spraying, coarsely mixing, tossing, kneading, pilling, and extruding oneor more unnatural isomer of reduced folate, singly or in combination,onto or with the nutritional substance.

One or more of the one or more natural isomers of reduced folate thatare added to the nutritional substance in accordance with the presentinvention can be substantially chirally pure or each of the one or morenatural isomers of reduced folate can be chirally pure. Alternatively,one or more of the one or more natural isomers of reduced folate can bepresent in a mixture with one or more unnatural isomers of reducedfolate. The molar amount of the one or more natural isomers of reducedfolate and the one or more unnatural isomers of reduced folate presentin the mixture added to the nutritional substance can be the same, as inthe case where a racemic mixture is added, or they can be different.Preferably the molar amount of the natural isomer exceeds the molaramount of the unnatural isomer. Additionally or alternatively, unnaturalisomer of reduced folate can be incorporated in a separate stepsubsequent to or prior to incorporating the one or more natural isomersof reduced folate into the nutritional substance. It is preferred thatthe collective molar amount of unnatural isomer of reduced folate addedbefore, during, and/or after the addition of natural isomer of reducedfolate be less than the collective molar amount of natural isomer added.

Natural isomers of reduced folates that are substantially chirally purecan be prepared by any suitable method, including, for example, by themethod described in U.S. Pat. No. 5,350,851 to Bailey et al., which ishereby incorporated by reference.

When the nutritional substance is a food preparation, in addition toincorporating a natural isomer of reduced folate, one or more essentialnutrients, optionally, can be incorporated into the food preparation.The essential nutrients can, for example, be added to the foodpreparation before, during, or after addition of the natural isomer ofreduced folate.

The compositions of the present invention can be used to increase asubject's dietary intake of folate by administering the composition tothe subject. The subject can be an animal, such as a dog or a cat;alternatively, the subject can be a human. Certain classes ofindividuals are viewed to be especially benefitted by increasing dietaryintake of folate. These include pregnant females; females who have had amiscarriage; females who have carried a fetus having a neural tubedefect, a cleft lip defect, or a cleft palate defect; and humans whosuffer vascular disease.

The compositions can also be used to treat a subject afflicted withintestinal malabsorption, especially folate malabsorption. When treatinga subject afflicted with intestinal malabsorption, the amount ofcomposition administered is preferably effective to cause an increase inthe subject's blood folate level. More preferably, the amountadministered is effective to produce blood folate levels in an normalrange, as determined by conventional blood-folate analysis methods, suchas with the Quanta Phase II assay from BioRad Laboratories, Hercules,Calif.

The compositions can be administered enterally, such as orally,intragastricly, or transpyloricly. Many factors that may modify theaction of the composition can be taken into account by those skilled inthe art; e.g., body weight, sex, diet, time of administration, route ofadministration, rate of excretion, condition of the subject, andreaction sensitivities and severities. Administration can be carried outcontinuously or periodically, such as once daily, or once with everymeal.

Compositions containing the natural isomer of reduced folates arepreferably for enteral consumption (enteral including oral,intragastric, or transpyloric), and are of any, food preparation, foodsupplement, essential nutrient preparation, or vitamin preparation.Typical examples of such food or vitamin preparations are those to whichfolic acid (I) is currently added for use by either humans or otheranimals. Natural isomer means a tetrahydrofolate having the naturalconfiguration at both the glutamate α- and the pteridine 6-carbons. Morespecifically, these compositions are, but are not limited to,multivitamin (with or without minerals and other nutrients) preparations(such preparations can be in solid, liquid or suspension forms);breakfast foods such as prepared cereals, breakfast drink mixes, toasterpastries and breakfast bars; infant formulas; dietary supplements andcomplete diet and weight-loss formulas and bars; animal feed or animalfeed supplements (for example, for poultry), and pet foods. The naturalisomer of reduced folates can also be used in products which healthofficials might in the future deem an appropriate vehicles forsatisfaction of the daily folate requirement. The composition of thepresent invention can include a natural isomer of a tetrahydrofolate,such as II--VIII, preferably the monoglutamate form, in a vitaminformulation preferably containing at least one other vitamin (forexample another vitamin such as a B vitamin) other than ascorbic acid(vitamin C), although ascorbic acid can be an additional component. Suchnutrients or vitamins can be those intended for either human or animaluse. Other ingredients may also be present, such as fillers, bindingagents, stabilizers, sweeteners, including nutitive sweeteners (e.g.sucrose, sorbitol and other polyols) and non-nutritive sweeteners (e.g.saccharin, aspartame, and acesulfame K), colorants, flavors, buffers,salts, coatings, and the like that are known to those skilled in the artof vitamin formulation.

For many applications of the described reduced folates (II through VIII)5-methyl-(6S)-tetrahydrofolic acid (III), 5-formyl-(6S)-tetrahydrofolicacid (IV), and 5,10-methenyl-(6R)-tetrahydrofolic acid (VII) arepreferred. All of the reduced folates are to differing extent subject tooxidation by air. Several factors influence this susceptibility,foremost being derivatization of the 5-nitrogen which considerably slowsoxidation. The 5-methyl- and 5-formyl derivatives are the most abundantforms of folate found in most tissues of the body. The predominatefolate in human blood is 5-methyl-(6S)-tetrahydrofolic acid (III).

In using tetrahydrofolates for increasing the folate in a nutritionalcomposition, several factors should be considered. Many nutrients areunstable to processing, including several of the vitamins. For example,vitamins A, B₁ (thiamin), and ascorbic acid are especially labile undersome conditions. Many procedures are known for enhancing the stabilityof the various nutrients such as pH and moisture control of thecomposition. Components which are to a degree incompatible with eachother, for example ascorbic acid with the more oxidized forms of iron orcopper, can be made to be present heterogeneously in the composition.Nutrients which are unstable to heat are often added after baking steps;for example, vitamins are often sprayed onto breakfast cerealssubsequent to toasting. Nutrients which are unstable to air can bepackaged in a reduced oxygen condition, and/or in containers that havelow or no permeability to oxygen. These and other procedures known tothose skilled in the art are useful for maintaining folates in theirnatural reduced form.

The rate of oxidation of a reduced folate in the presence of air isincreased in water solutions. The shelf life of liquid preparations willbe greatly extended if they are preferably kept in air-tight containers.Replacing air with an inert gas such as nitrogen or argon also retardsloss. Reduced folates can also be protected from oxidation by a numberof reducing agents and antioxidants, the most relevant of these beingother vitamins that are often included in multivitamin mixtures ornutritional compositions. Ascorbic acid has been used for protection ofreduced folates in biochemical experiments and procedures for thelaboratory analysis of biological samples. Such protection need not belimited to use of ascorbic acid or other vitamins; other agents suitablefor human or animal consumption are useful, for example iso-ascorbicacid and certain thiols, such as glutathione. Further, known packagingand formulation technologies which increase the stability of compoundssuch as ascorbic acid or other air labile materials (for example, coatedforms, blister packaging, and use of reduced metals or metal complexes)are useful for the maintenance of reduced folates. The salt form of areduced folate also somewhat affects stability and solubility, and thiscan be optimized for the needs of each product. The pH of the finalcomposition can also be optimized according to the stability propertiesof the particular reduced folate derivative used and of the othercomponents present, as is well understood in the arts of processingnutrients and of folate compounds. For example, in the presence ofmoisture 5,10-methenyl-(6R)-FH₄ (VII) can be transformed into10-formyl-(6R)-FH₄ and 5-formyl-(6S)-FH₄ (IV) (the latter also apreferred compound) in a pH dependent manner. Compostions containing5,10-methenyl-(6R)-FH₄ (VII) are most stable to oxidation when eithersubstantially dry and/or have an acidity less than about pH 4. Withproper attention to the above factors, the lability of reduced folatesneed not limit the life of a product, especially with III and IV whichare more resistant than ascorbic acid to many oxidation reactions.

The substitution of a reduced folate for folic acid (I) should take intoaccount the differences in molecular weights of the various forms. Forexample, the current U.S. Reference Daily Intake of 0.4 mg of folic acid(I) corresponds to 0.91 micromole using an anhydrous molecular weight of441.4. The effective molecular weight of reduced folates depends uponthe derivative employed (i.e. II-VIII), the salt form, and watercontent. For example, 0.91 micromole of 5-formyl-(6S)-tetrahydrofolicacid (IV) calcium salt-pentahydrate would weigh 0.545 mg, and 0.91micromole of 5-methyl-(6S)-tetrahydrofolic acid (III) disodium saltwould weigh 0.456 mg. Several salt forms of the reduced folates aredescribed in the literature, such as hydrochloride, sodium, potassium,magnesium, calcium, and others and having various water content. Foreach of these forms a similar calculation can be made. The amountrequired to achieve the mole equivalent to a desired fraction of the RDIwould then be that fraction of this new weight. As an example, 25% RDIof 5-methyl-(6S)-tetrahydrofolic acid (III) disodium salt would be0.25×0.456=0.114 mg, the mole equivalent of 0.10 mg of folic acid (I).Previous investigations of groups of individuals having a normal uptakeof folic acid (I) have shown that the bioavailability of the reducedfolates is similar on a mole basis.

As mentioned above, loss of nutrients during processing, especially offoods is well known to those skilled in this art. An often practicedprocedure is the addition of an initial excess, an "overage", of aparticular nutrient or nutrients, such that the final post-processingamount is at the desired level. Many highly sensitive and specificmethods are known (such as microbial growth dependence, folate bindingprotein based assays, HPLC and GC) for the analysis of folates, in boththe reduced and oxidized forms as well as for their various derivatives.These assays permit adjustment of the added amount of the natural isomerof a reduced folate so as to yield the desired final amount subsequentto processing and packaging. The range of the natural isomer of areduced folate in the composition of this invention is preferably thatfulfilling between about 5% and about 200% of the RDI of humans forfolate, and should be taken to encompass both the situation whereallowance is made for processing loss, and also where no such allowanceis made. Separate RDA dosages are specified for different groups ofpeople, for example pregnant and non-pregnant women. Further, the RDIlevel although relying on RDA values, can be different from RDA values.The above range of "between about 5% and about 200% of the RDI forfolate" should be taken to operate independently on each of theseseparate RDA and RDI specifications, or their foreign equivalents, aspresemtly stated or as modified in the future. For the purpose of thisinvention these several specifications shall be referred to as the dailyrequirement for folate. Unless the recommended dietary allowance forfolate in humans is increased, the maximum final amount of the naturalisomer of a reduced folate in composition for human use in satisfyingthe daily requirement for folate preferably should not exceed about 4.5micromole per dose or customarily consumed serving. However, forindividuals afflicted with intestinal malabsorption, such as celiacdisease or tropical sprue, compositions containing higher amounts of thenatural isomer of a reduced folate will be useful.

For the purpose of this invention an essential nutrient composition canbe a dietary supplement or the like, the substantial folate component ofwhich is derived from substantially pure tetrahydrofolic acid orderivative thereof, such as compounds II through VIII. Essentialnutrient compositions encompassed by this invention comprise the naturalisomer of a reduced folate preferably within the above described rangealong with other vitamins and/or other nutrients which are preferablyeach present in an amount that is considered to be safe. In formulatingcompositions for animal consumption manufacturers often considerablyexceed the dosage recommended by the NRC for folate (by 10-fold,20-fold, or more in some cases), not only to overcome losses duringprocessing, but also to cover occasions of possible increased need forfolate, such as during antibiotic treatment. Other vitamin and nutrientcomponents can be present in amounts that vary considerably from NRCrecommendations. The following examples are given to further illustratethe invention, and are not intended to limit its scope in any way.

EXAMPLES

1) A typical ready to eat breakfast cereal: corn (and/or other grains),sugar, salt, malt flavoring, such that a 30 g serving provides about 2 gof protein, 26 g total carbohydrate, and 330 mg of sodium, alsocontaining per serving size vitamin A palmitate (15% of RDI), ascorbicacid (25% of RDI), reduced iron (45% of RDI), vitamin D (10% of RDI),thiamin hydrochloride (25% of RDI), riboflavin (25% of RDI), niacinamide(25% of RDI), pyridoxine hydrochloride (25% of RDI), and 0.114 mg of5-methyl-6(S)-tetrahydrofolic acid (III) disodium salt (the moleequivalent of 0.1 mg folic acid, 25% of RDI).

2) A typical daily multivitamin tablet: calcium carbonate, ascorbic acid(60 mg, 100% RDI), gelatin, vitamin E acetate (30 I.U., 100% RDI),starch, niacinamide (20 mg, 100% RDI), hydroxypropyl-methylcellulose,calcium pantothenate (10 mg, 100% RDI), calcium silicate,hydroxypropylcellulose, pyridoxine hydrochloride (2 mg, 100% RDI),riboflavin (1.7 mg, 100% RDI), thiamin mononitrate (1.5 mg, 100% RDI),beta carotene & vitamin A acetate (5000 I.U., 100% RDI), sodiumhexametaphosphate, magnesium stearate, vitamin D (400 I.U., 100% RDI),vitamin B,₂ (6 μg, 100% RDI), lecithin, and 0.437 mg of5-methyl-6(S)-tetrahydrofolic acid (III) magnesium salt (the moleequivalent of 0.4 mg folic acid, 100% of RDI).

3) A typical daily multivitamin and minerals tablet: calcium phosphate(130 mg of elemental calcium), magnesium hydroxide & stearate (100 mg,25% RDI), cellulose, potassium chloride, ascorbic acid (60 mg, 100%RDI), gelatin,ferrous fumarate (18 mg elemental iron, 100% RDI), zincsulfate (15 mg, 100% RDI), modified cellulose gum, vitamin E acetate (30I.U., 100% RDI), citric acid, niacinamide (20 mg, 100% RDI), magnesiumstearate, hydroxypropyl-methylcellulose, calcium pantothenate (10 mg,100% RDI), selenium yeast, polyvinylpyrrolidone, hydroxypropylcellulose,manganese sulfate, silica, copper oxide (2 mg, 100% RDI), chromiumyeast, molybdenum yeast, pyridoxine hydrochloride (2 mg, 100% RDI),riboflavin (1.7 mg, 100% RDI), thiamin mononitrate (1.5 mg, 100% RDI),beta carotene & vitamin A acetate (5000 I.U., 100% RDI), potassiumiodide (150 μg, 100% RDI), sodium hexametaphosphate, biotin (30 μg, 10%RDI), vitamin D (400 I.U., 100% RDI), vitamin B₁₂ (6 μg, 100% RDI),lecithin, and 0.545 mg 5-formyl-(6S)-tetrahydrofolic acid (IV) calciumsalt-pentahydrate (the mole equivalent of 0.4 mg of folic acid, 100%RDI).

4) A typical daily multivitamin and minerals tablet for older adults:calcium carbonate, calcium phosphate (200 mg Ca, 20% RDI; 48 mgphosphorous, 5% RDI), magnesium oxide, magnesium stearate (100 mg, 25%RDI), potassium chloride (80 mg, 2% RDI), microrystalline cellulose,ascorbic acid (60 mg, 100% RDI), gelatin, d l-alfa-tocopheryl acetate(45 I.U., 150% RDI), modified food starch, maltodextrin, crospovidone,reduced iron (4 mg, 22 RDI), hydroxypropyl methylcellulose, niacinamide(20 mg, 100% RDI), zinc oxide (15 mg, 100% RDI), calcium pantothenate,manganese sulfate (3.5 mg), vitamin D (400 I.U., 100% RDI), titaniumdioxide, vitamin A and β-carotene (5000 I.U., 100% RDI), stearic acid,pyridoxine hyrochloride (3 mg, 150% RDI), riboflavin (1.7 mg, 100% RDI),silicon dioxide, copper oxide (2 mg, 100% RDI), dextrose, thiaminmononitrate (1.5 mg, 100% RDI), triethyl citrate, polysorbate 80,chhromium chloride (130 μg), artificial colors, potassium iodide ((150μg, 100% RDI), sodium metasilicate (2 mg), sodium molybdate (160 μg),borates, sodium selenate (20 μg), biotin (30 μg, 10% RDI), sodiummetavanadate (10 μg), cyanocobalamin (25 μg, 417% RDI), nickeloussulfate (5 μg), and phytonadione, and 5,10-methenyl-(6R)-tetrahydrofolicacid hydrochloride (VII)(0.44 mg, the mole equivalent of 0.4 mg of folicacid, 100% RDI).

5) A typical complete diet drink: water, sugar, calcium and sodiumcaseinates, maltodextrin, high-oleic safflower oil, soy protein, soyoil, canola oil, cocoa, sodium and potassium citrates, calcium carbonateand phosphate (250 mg Ca, 25% RDI), magnesium chloride and phosphate(100 mg Mg, 25% RDI), sodium chloride, soy lecithin, choline chloride,flavor, ascorbic acid (30 mg, 50% RDI), carrageenan, zinc sulfate (5.6mg, 37% RDI), ferrous sulfate (4.5 mg Fe, 25% RDI), alfa-tocopherylacetate (11.3 I.U., 37.7% RDI), niacinamide (5 mg, 25% RDI), calciumpantothenate (2.5 mg, 25% RDI), manganese sulfate (1.3 mg), copper salt(25% RDI), vitamin A palmitate (1250 I.U., 25% RDI), thiaminhydrochloride (0.375 mg, 25% RDI), pyridoxine hydrochloride (0.5 mg, 25%RDI), riboflavin (0.425 mg, 25% RDI), biotin (75 μg, 25% RDI), sodiummolybdate (38 μg), chromium chloride (25 μg), potassium iodide (37.5 μg,25% RDI), sodium selenate (18 μg), phylloquinone (vitamin K₁),cyanocobalamin (1.5 μg, 25% RDI), vitamin D₃ (100 I.U., 25% RDI), and0.136 mg 5-formyl-(6S)-tetrahydrofolic acid (IV) calciumsalt-pentahydrate (the mole equivalent of 0.1 mg of folic acid, 25%RDI), packaged in an air-tight container, and supplying about 225calories.

6) A typical enhanced B-vitamin/tetrahydrofolate tablet: dibasic calciumphosphate, pyridoxine hydrochloride (50 mg, 2,500% RDI), cellulose,stearic acid, magnesium stearate, and 0.912 mg of5-methyl-6(S)-tetrahydrofolic acid (III) disodium salt (the moleequivalent of 0.8 mg folic acid, 200% of RDI for adults, 100% RDA forpregnant women).

7) A typical poultry feed vitamin supplement: (amounts per kg of diet)vitamin A (trans retinyl acetate, 5500 I.U.), vitamin E (11 I.U.),menadione sodium bisulfite (1.1 mg), vitamin D₃ (1100 I.U.), riboflavin(4.4 mg), vitamin B₁₂ (10 μg), vitamin B₆ (3.0 mg), thiamin mononitrate(2.2 mg), biotin (0.3 mg), ethoxyquin (125 mg), and 2.0 mg5-formyl-(6S)-tetrahydrofolic acid (IV) calcium salt-pentahydrate (themole equivalent of 1.45 mg of folic acid).

8) A typical dry cat food: ground yellow corn, corn gluten meal, soybeanmeal, poultry by-product meal, animal fat, fish meal, meat and bonemeal, ground wheat, phosphoric acid calcium carbonate, dried animaldigest, salt, brewers dried yeast, potassium chloride, dried wheysolubles, choline chloride, dried skimmed milk, taurine, L-lysine, zincoxide, ferrous sulfate, niacin, vitamin A, vitamin D₃, vitamin B₁₂,calcium pantothenate, citric acid, manganese sulfate, riboflavinsupplement, biotin, copper salt, thiamine mononitrate, pyridoxinehydrochloride, menadione sodium bisulfate complex, such that the crudeprotein is not less than 31%, crude fat is not less than 8%, crude fiberis not more than 4.5%, moisture is not more than 12%, calcium is notless than 1.2%, phosphorous is not less than 1.0%, sodium chloride isnot more than 1.5%, the metabolizable energy is about 3,600 kcal/kg,taurine, iron, vitamins A, D₃, B₁₂, and E are at least 100% of levelsrecommended by the Association of American Feed Control Officials, andcontaining not less than 0.97 mg/kg diet 5-methyl-6(S)-tetrahydrofolicacid (III) calcium salt dihydrate (the mole equivalent of 0.8 mg/kg dietof folic acid).

9) A typical soy based infant formula: 75.5% water; 13% sucrose; 6.6%oleo oil: coconut, high oleic (safflower or sunflower), and soybeanoils; 3.8% soy protein isolate; (protein 2.7 g, fat 5.3 g, carbohydrate10.2, linoleic acid 500 mg); potassium citrate and bicarbonate(potassium 105 mg); monobasic potassium and dibasic calcium phosphates(phosphorous 63 mg); soy lecithin; taurine; calcium carrageenan; calciumhydroxide, chloride and citrate (calcium 90 mg); sodium chloride (sodium30 mg); L-methionine; zinc (Zn 0.8 mg), ferrous (Fe 1.8 mg), andmanganese (Mn 30 μg) sulfates; copper salt (Cu 70 μg); taurine;L-carmiting; potassium iodide (I 9 μg); ascorbic acid (8.3 mg); cholinechloride; alpha-tocopheryl acetate (1.4 I.U.); niacinamide (750 μg);vitamin A palmitate and beta-carotene (300 I.U.); calcium pantothenate(450 μg); thiamin hydrochloride (100 μg); riboflavin (150 μg);pyridoxine hydrochloride (62.5 μg); vitamin K₁ (15 μg); biotin (5.5 μg);vitamin D₃ (60 I.U.); cyanocobalamin (0.3 μg); and 9.1 μg of5-methyl-6(S)-tetrahydrofolic acid (III) calcium salt dihydrate (themole equivalent of 7.5 μg of folic acid), packaged in an air-tightcontainer (amounts are per 150 ml of 1:1 diluted formula).

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned. While the above description contains manyspecificities, these should not be construed as limitations on the scopeof the invention, but rather as an exemplification of preferredembodiments thereof. Changes therein and other uses will occur to thoseskilled in the art which are encompassed within the spirit of theinvention as defined by the scope of the claims and their legalequivalents.

We claim:
 1. A composition for human consumption comprising:one or morenatural isomers of reduced folate selected from the group consisting of(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; and a nutritional substance for human consumption selected fromthe group consisting of a food preparation, an essential nutrientpreparation, and combinations thereof; wherein the total amount of saidone or more natural isomers of reduced folate in said composition isbetween about 5% and about 200% of a human daily requirement for folateper customarily consumed quantity of said composition; wherein, when thenutritional substance is a food preparation, the food preparationcomprises two or more food components and each gram of said foodpreparation has a natural molar amount, N, of said one or more naturalisomers of reduced folate, wherein N is greater or equal to zero andwherein each gram of said composition has a total molar amount, T, ofsaid one or more natural isomers of reduced folate greater than N;wherein, when the nutritional substance is an essential nutrientpreparation, the essential nutrient preparation comprises a vitaminother than ascorbic acid; and wherein, when the nutritional substance isan essential nutrient preparation and when said composition comprises anamount of 5-formyl-(6S)-tetrahydrofolic acid, said composition furthercomprises no 5-formyl-(6R)-tetrahydrofolic acid, or, if present, saidcomposition further comprises 5-formyl-(6R)-tetrahydrofolic acid in anamount less than the amount of 5-formyl-(6S)-tetrahydrofolic acidpresent in said composition.
 2. A composition for human consumptioncomprising:one or more natural isomers of reduced folate selected fromthe group consisting of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; and a nutritional substance for human consumption selected fromthe group consisting of a food preparation, an essential nutrientpreparation, and combinations thereof; wherein the total amount of saidone or more natural isomers of reduced folate in said composition isless than or equal to about 4.5 micromoles per customarily consumedquantity of said composition; wherein, when the nutritional substance isa food preparation, the food preparation comprises two or more foodcomponents and each gram of said food preparation has a natural molaramount, N, of said one or more natural isomers of reduced folate,wherein N is greater or equal to zero and wherein each gram of saidcomposition has a total molar amount, T, of said one or more naturalisomers of reduced folate greater than N; wherein, when the nutritionalsubstance is an essential nutrient preparation, the essential nutrientpreparation comprises a vitamin other than ascorbic acid; and wherein,when the nutritional substance is an essential nutrient preparation andwhen said composition comprises an amount of5-formyl-(6S)-tetrahydrofolic acid, said composition further comprisesno 5-formyl-(6R)-tetrahydrofolic acid, or, if present, said compositionfurther comprises 5-formyl-(6R)-tetrahydrofolic acid in an amount lessthan the amount of 5-formyl-(6S)-tetrahydrofolic acid present in saidcomposition.
 3. A composition according to claim 1 or 2, wherein theessential nutrient preparation further comprises ascorbic acid.
 4. Acomposition according to claim 1 or 2, wherein the one or more naturalisomers of reduced folate is selected from the group consisting of5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof.
 5. A composition according to claim 4, wherein the one or morenatural isomers of reduced folate is 5-methyl-(6S)-tetrahydrofolic acidor a polyglutamyl derivative thereof.
 6. A composition according toclaim 4, wherein the one or more natural isomers of reduced folate is5-formyl-(6S)-tetrahydrofolic acid or a polyglutamyl derivative thereof.7. A composition according to claim 4, wherein the one or more naturalisomers of reduced folate is 5,10-methenyl-(6R)-tetrahydrofolic acid ora polyglutamyl derivative thereof.
 8. A composition according to claim 1or 2, wherein each of the one or more natural isomers of reduced folateis substantially chirally pure.
 9. A method for increasing the folatecontent of a nutritional substance for human consumptioncomprising:providing a nutritional substance for human consumptionselected from the group consisting of a food preparation, an essentialnutrient preparation, and combinations thereof; and incorporating intothe nutritional substance an amount of one or more natural isomers ofreduced folate selected from the group consisting of(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof, thereby forming a composition; wherein the total amount of saidone or more natural isomers of reduced folate incorporated into saidnutritional substance is between about 5% and about 200% of a humandaily requirement for folate per customarily consumed quantity of saidnutritional substance; wherein, when the nutritional substance is a foodpreparation, the food preparation comprises two or more food componentsand each gram of said food preparation has a natural molar amount, N, ofsaid one or more natural isomers of reduced folate, wherein N is greateror equal to zero and wherein each gram of said composition has a totalmolar amount, T, of said one or more natural isomers of reduced folategreater than N; wherein, when the nutritional substance is an essentialnutrient preparation, the essential nutrient preparation comprises avitamin other than ascorbic acid; and wherein, when the nutritionalsubstance is an essential nutrient preparation and when the compositioncomprises an amount of 5-formyl-(6S)-tetrahydrofolic acid, thecomposition further comprises no 5-formyl-(6R)-tetrahydrofolic acid, or,if present, the composition further comprises5-formyl-(6R)-tetrahydrofolic acid in an amount less than the amount of5-formyl-(6S)-tetrahydrofolic acid present in the composition.
 10. Amethod for increasing the folate content of a nutritional substance forhuman consumption comprising:providing a nutritional substance for humanconsumption selected from the group consisting of a food preparation, anessential nutrient preparation, and combinations thereof; andincorporating into the nutritional substance an amount of one or morenatural isomers of reduced folate selected from the group consisting of(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof, thereby forming a composition; wherein the total amount of saidone or more natural isomers of reduced folate in said composition isless than or equal to about 4.5 micromoles per customarily consumedquantity of said composition; wherein, when the nutritional substance isa food preparation, the food preparation comprises two or more foodcomponents and each gram of said food preparation has a natural molaramount, N, of said one or more natural isomers of reduced folate,wherein N is greater or equal to zero and wherein each gram of saidcomposition has a total molar amount, T, of said one or more naturalisomers of reduced folate greater than N; wherein, when the nutritionalsubstance is an essential nutrient preparation, the essential nutrientpreparation comprises a vitamin other than ascorbic acid; and wherein,when the nutritional substance is an essential nutrient preparation andwhen the composition comprises an amount of5-formyl-(6S)-tetrahydrofolic acid, the composition further comprises no5-formyl-(6R)-tetrahydrofolic acid, or, if present, the compositionfurther comprises 5-formyl-(6R)-tetrahydrofolic acid in an amount lessthan the amount of 5-formyl-(6S)-tetrahydrofolic acid present in thecomposition.
 11. A method according to claim 9 or 10, wherein the one ormore natural isomers of reduced folate is selected from the groupconsisting of 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolicacid, and polyglutamyl derivatives thereof.
 12. A method according toclaim 11, wherein the one or more natural isomers of reduced folate is5-methyl-(6S)-tetrahydrofolic acid or a polyglutamyl derivative thereof.13. A method according to claim 11, wherein the one or more naturalisomers of reduced folate is 5-formyl-(6S)-tetrahydrofolic acid or apolyglutamyl derivative thereof.
 14. A method according to claim 11,wherein the one or more natural isomers of reduced folate is5,10-methenyl-(6R)-tetrahydrofolic acid or a polyglutamyl derivativethereof.
 15. A method according to claim 10 wherein said method furthercomprises:incorporating a vitamin into the food preparation.
 16. Amethod according to claim 9 or 10, further comprising:incorporating intothe nutritional substance an amount of one or more unnatural isomers ofreduced folate selected from the group consisting of(6R)-tetrahydrofolic acid, 5-methyl-(6R)-tetrahydrofolic acid,5-formyl-(6R)-tetrahydrofolic acid, 10-formyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6S)-tetrahydrofolic acid,5,10-methenyl-(6S)-tetrahydrofolic acid,5-formimino-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof, wherein the amount of the one or more unnatural isomers ofreduced folate is less than or equal to the amount of the one or morenatural isomers of reduced folate.
 17. A method according to claim 9 or10, wherein the essential nutrient preparation further comprisesascorbic acid.
 18. A method according to claim 9 or 10, wherein each ofthe one or more natural isomers of reduced folate is substantiallychirally pure.
 19. A composition for consumption by an animal, theanimal selected from the group consisting of dog, cat, poultry, cattle,goat, horse, mink, fox, and sheep, the composition comprising:one ormore natural isomers of reduced folate selected from the groupconsisting of (6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolicacid, 5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolicacid, 5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; and a nutritional substance for consumption by the animalselected from the group consisting of a food preparation, an essentialnutrient preparation, and combinations thereof; wherein the total amountof said one or more natural isomers of reduced folate in saidcomposition is between about 5% and about 3000% of an animal dailyrequirement for folate per customarily consumed quantity of saidcomposition; wherein, when the nutritional substance is a foodpreparation, the food preparation comprises two or more food componentsand each gram of said food preparation has a natural molar amount, N, ofsaid one or more natural isomers of reduced folate, wherein N is greateror equal to zero and wherein each gram of said composition has a totalmolar amount, T, of said one or more natural isomers of reduced folategreater than N; wherein, when the nutritional substance is an essentialnutrient preparation, the essential nutrient preparation comprises avitamin other than ascorbic acid; and wherein, when the nutritionalsubstance is an essential nutrient preparation and when said compositioncomprises an amount of 5-formyl-(6S)-tetrahydrofolic acid, saidcomposition further comprises no 5-formyl-(6R)-tetrahydrofolic acid, or,if present, said composition further comprises5-formyl-(6R)-tetrahydrofolic acid in an amount less than the amount of5-formyl-(6S)-tetrahydrofolic acid present in said composition.
 20. Acomposition according to claim 19, wherein the essential nutrientpreparation further comprises ascorbic acid.
 21. A composition accordingto claim 19, wherein the one or more natural isomers of reduced folateis selected from the group consisting of 5-methyl-(6S)-tetrahydrofolicacid, 5-formyl-(6S)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof.
 22. A composition according to claim 21, wherein the one ormore natural isomers of reduced folate is 5-methyl-(6S)-tetrahydrofolicacid or a polyglutamyl derivative thereof.
 23. A composition accordingto claim 21, wherein the one or more natural isomers of reduced folateis 5-formyl-(6S)-tetrahydrofolic acid or a polyglutamyl derivativethereof.
 24. A composition according to claim 21, wherein the one ormore natural isomers of reduced folate is5,10-methenyl-(6R)-tetrahydrofolic acid or a polyglutamyl derivativethereof.
 25. A composition according to claim 19, wherein each of theone or more natural isomers of reduced folate is substantially chirallypure.
 26. A method for increasing the folate content of a nutritionalsubstance for consumption by an animal, the animal selected from thegroup consisting of dog, cat, poultry, cattle, goat, horse, mink, fox,and sheep, the method comprising:providing a nutritional substance forconsumption by the animal selected from the group consisting of a foodpreparation, an essential nutrient preparation, and combinationsthereof; and incorporating into the nutritional substance an amount ofone or more natural isomers of reduced folate selected from the groupconsisting of (6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolicacid, 5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolicacid, 5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof, thereby forming a composition; wherein the total amount of saidone or more natural isomers of reduced folate incorporated into saidnutritional substance is between about 5% and about 3000% of an animaldaily requirement for folate per customarily consumed quantity of saidnutritional substance; wherein, when the nutritional substance is a foodpreparation, the food preparation comprises two or more food componentsand each gram of said food preparation has a natural molar amount, N, ofsaid one or more natural isomers of reduced folate, wherein N is greateror equal to zero and wherein each gram of said composition has a totalmolar amount, T, of said one or more natural isomers of reduced folategreater than N; wherein, when the nutritional substance is an essentialnutrient preparation, the essential nutrient preparation comprises avitamin other than ascorbic acid; and wherein, when the nutritionalsubstance is an essential nutrient preparation and when the compositioncomprises an amount of 5-formyl-(6S)-tetrahydrofolic acid, thecomposition further comprises no 5-formyl-(6R)-tetrahydrofolic acid, or,if present, the composition further comprises5-formyl-(6R)-tetrahydrofolic acid in an amount less than the amount of5-formyl-(6S)-tetrahydrofolic acid present in the composition.
 27. Amethod according to claim 26 further comprising:incorporating into thenutritional substance an amount of one or more unnatural isomers ofreduced folate selected from the group consisting of(6R)-tetrahydrofolic acid, 5-methyl-(6R)-tetrahydrofolic acid,5-formyl-(6R)-tetrahydrofolic acid, 10-formyl-(6S)-tetrahydrofolic acid,5,10-methylene-(6S)-tetrahydrofolic acid,5,10-methenyl-(6S)-tetrahydrofolic acid,5-formimino-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof, wherein the amount of the one or more unnatural isomers ofreduced folate is less than or equal to the amount of the one or morenatural isomers of reduced folate.
 28. A method according to claim 26,wherein the essential nutrient preparation further comprises ascorbicacid.
 29. A method according to claim 26, wherein each of the one ormore natural isomers of reduced folate is substantially chirally pure.30. A method according to claim 26, wherein the one or more naturalisomers of reduced folate is selected from the group consisting of5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof.
 31. A method according to claim 30, wherein the one or morenatural isomers of reduced folate is 5-methyl-(6S)-tetrahydrofolic acidor a polyglutamyl derivative thereof.
 32. A method according to claim30, wherein the one or more natural isomers of reduced folate is5-formyl-(6S)-tetrahydrofolic acid or a polyglutamyl derivative thereof.33. A method according to claim 30, wherein the one or more naturalisomers of reduced folate is 5,10-methenyl-(6R)-tetrahydrofolic acid ora polyglutamyl derivative thereof.
 34. A method according to claim 26,wherein said method further comprises:incorporating a vitamin into thefood preparation.
 35. A method of increasing a human subject's dietaryintake of folate comprising administering to the human subject acomposition comprising:one or more natural isomers of reduced folateselected from the group consisting of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; and a nutritional substance for human consumption selected fromthe group consisting of a food preparation, an essential nutrientpreparation, and combinations thereof; wherein the total amount of saidone or more natural isomers of reduced folate in said composition isbetween about 5% and about 200% of a human daily requirement for folateper customarily consumed quantity of said composition; wherein, when thenutritional substance is a food preparation, the food preparationcomprises two or more food components and each gram of said foodpreparation has a natural molar amount, N, of said one or more naturalisomers of reduced folate, wherein N is greater or equal to zero andwherein each gram of said composition has a total molar amount, T, ofsaid one or more natural isomers of reduced folate greater than N; andwherein, when the nutritional substance is an essential nutrientpreparation, the essential nutrient preparation comprises a vitaminother than ascorbic acid.
 36. A method of increasing a human subject'sdietary intake of folate comprising administering to the human subject acomposition for human consumption comprising:one or more natural isomersof reduced folate selected from the group consisting of(6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid,5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid,5,10-methylene-(6R)-tetrahydrofolic acid,5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; and a nutritional substance for human consumption being a foodpreparation; wherein the food preparation comprises two or more foodcomponents and each gram of said food preparation has a natural molaramount, N, of said one or more natural isomers of reduced folate,wherein N is greater or equal to zero and wherein each gram of saidcomposition has a total molar amount, T, of said one or more naturalisomers of reduced folate greater than N; and wherein each gram of saidcomposition further comprises no unnatural isomers of reduced folateselected from the group consisting of (6R)-tetrahydrofolic acid,5-methyl-(6R)-tetrahydrofolic acid, 5-formyl-(6R)-tetrahydrofolic acid,10-formyl-(6S)-tetrahydrofolic acid, 5,10-methylene-(6S)-tetrahydrofolicacid, 5,10-methenyl-(6S)-tetrahydrofolic acid,5-formimino-(6R)-tetrahydrofolic acid, and polyglutamyl derivativesthereof, or, if present, one or more of said unnatural isomers ofreduced folate in a molar amount less than T minus N.
 37. A method ofincreasing a human subject's dietary intake of folate comprisingadministering to the human subject a composition for human consumptioncomprising:one or more natural isomers of reduced folate selected fromthe group consisting of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; and a nutritional substance for human consumption being anessential nutrient preparation, the essential nutrient preparationcomprising a vitamin other than ascorbic acid, wherein the vitamin ispresent in an amount equal to or greater than 25% of the dailyrequirement for the vitamin per customarily consumed quantity of saidessential nutrient preparation.
 38. A method of increasing a humansubject's dietary intake of folate comprising administering to the humansubject a composition comprising:one or more natural isomers of reducedfolate selected from the group consisting of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; and a nutritional substance for human consumption selected fromthe group consisting of a food preparation, an essential nutrientpreparation, and combinations thereof; wherein the total amount of saidone or more natural isomers of reduced folate in said composition isless than or equal to about 4.5 micromoles per customarily consumedquantity of said composition; wherein, when the nutritional substance isa food preparation, the food preparation comprises two or more foodcomponents and each gram of said food preparation has a natural molaramount, N, of said one or more natural isomers of reduced folate,wherein N is greater or equal to zero and wherein each gram of saidcomposition has a total molar amount, T, of said one or more naturalisomers of reduced folate greater than N; and wherein, when thenutritional substance is an essential nutrient preparation, theessential nutrient preparation comprises a vitamin other than ascorbicacid.
 39. A method according to claim 35, 36, 37, or 38, wherein saidadministering is carried out by enteral administration.
 40. A methodaccording to claim 35, 36, 37, or 38, wherein the human is selected fromthe group consisting of a pregnant female; a female who has had amiscarriage; a female who has carried a fetus having a neural tubedefect, a cleft lip defect, or a cleft palate defect; and a human whosuffers vascular disease.
 41. A method according to claim 35, 37, or 38,wherein, when the nutritional substance is an essential nutrientpreparation and when said composition comprises an amount of5-formyl-(6S)-tetrahydrofolic acid, said composition further comprisesno 5-formyl-(6R)-tetrahydrofolic acid, or, if present, said compositionfurther comprises 5-formyl-(6R)-tetrahydrofolic acid in an amount lessthan the amount of 5-formyl-(6S)-tetrahydrofolic acid present in saidcomposition.
 42. A method of increasing an animal subject's dietaryintake of folate, the animal selected from the group consisting of dog,cat, poultry, cattle, goat, horse, mink, fox, and sheep, the methodcomprising administering to the animal subject a compositioncomprising:one or more natural isomers of reduced folate selected fromthe group consisting of (6S)-tetrahydrofolic acid,5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid,10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolicacid, 5,10-methenyl-(6R)-tetrahydrofolic acid,5-formimino-(6S)-tetrahydrofolic acid, and polyglutamyl derivativesthereof; and a nutritional substance for consumption by the animalselected from the group consisting of a food preparation, an essentialnutrient preparation, and combinations thereof; wherein the total amountof said one or more natural isomers of reduced folate in saidcomposition is between about 5% and about 3000% of an animal dailyrequirement for folate per customarily consumed quantity of saidcomposition; wherein, when the nutritional substance is a foodpreparation, the food preparation comprises two or more food componentsand each gram of said food preparation has a natural molar amount, N, ofsaid one or more natural isomers of reduced folate, wherein N is greateror equal to zero and wherein each gram of said composition has a totalmolar amount, T, of said one or more natural isomers of reduced folategreater than N; and wherein, when the nutritional substance is anessential nutrient preparation, the essential nutrient preparationcomprises a vitamin other than ascorbic acid.
 43. A method according toclaim 42, wherein said administering is carried out by enteraladministration.
 44. A method according to claim 42, wherein, when thenutritional substance is an essential nutrient preparation and when saidcomposition comprises an amount of 5-formyl-(6S)-tetrahydrofolic acid,said composition further comprises no 5-formyl-(6R)-tetrahydrofolicacid, or, if present, said composition further comprises5-formyl-(6R)-tetrahydrofolic acid in an amount less than the amount of5-formyl-(6S)-tetrahydrofolic acid present in said composition.